New Role of the Disulfide Stress Effector YjbH in β-Lactam Susceptibility of Staphylococcus aureus

Abstract

Staphylococcus aureus is exposed to multiple antimicrobial compounds, including oxidative burst products and antibiotics. The various mechanisms and regulatory pathways governing susceptibility or resistance are complex and only superficially understood. Bacillus subtilis recently has been shown to control disulfide stress responses by the thioredoxin-related YjbH protein, which binds to the transcriptional regulator Spx and controls its degradation via the proteasome-like ClpXP protease. We show that the S. aureus YjbH homolog has a role in susceptibility to the disulfide stress-inducing agent diamide that is similar to that in B. subtilis, and we demonstrate that the four cysteine residues in YjbH are required for this activity. In addition, the inactivation of YjbH led to moderate resistance to oxacillin and other ␤-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan crosslinking. Of note, the impact of YjbH on ␤-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and ␤-lactam resistance rely on different types of interactions. These data suggest that the ClpXP adaptor YjbH has more target proteins than previously thought, and that oxidative burst and ␤-lactam resistance mechanisms of S. aureus are closely linked.Staphylococcus aureus is a major human pathogen causing a wide spectrum of diseases that range from mild skin infections to life-threatening septicemia, pneumonia, and toxic-shock syndrome (27). ␤-Lactam antibiotics such as oxacillin are among to the most effective drugs against S. aureus infections, but their usefulness is continuously decreasing because ␤-lactam-resistant strains, particularly methicillin-resistant S. aureus (MRSA), are spreading in hospitals and, more recently, in the community at large, alarming international health authorities (11). Resistance is based on ␤-lactamases (penicillin-resistant S. aureus) or on an alternative peptidoglycan-biosynthetic enzyme, the penicillin-binding protein 2a (MRSA) (5, 14). In addition, mutations in a variety of proteins affecting cell wall biosynthesis and turnover can affect ␤-lactam susceptibility, leading either to hypersensitivity or to moderate levels of resistance (3,5,9,17,24,36). In many cases it remains unclear how the mutated proteins affect staphylococcal ␤-lactam resistance, and the underlying mechanisms remain only superficially understood.During the process of infection, S. aureus is engulfed by phagocytic cells such as neutrophils and macrophages, and the bacteria are exposed to the microbicidal activity of these cells.One of the most powerful antimicrobial mechanisms is based on the production of reactive oxygen species (ROS) or nitrogen species (RNS) by phagocyte NADPH oxidase and nitric oxide synthase, respectively (19,29). In addition, ROS also are generated during incomplete electron transfer in the bacterial respiratory chain (21). Therefore, even nonpath...

The YjbH Adaptor Protein Enhances Proteolysis of the Transcriptional Regulator Spx in Staphylococcus aureus

“…The cysteine residues were replaced with glycine residues, and plasmid-encoded cysteine replacement mutants of YjbH were tested for their ability to complement an S. aureus yjbH mutant strain. In contrast to our results, Göhring et al (12) suggest that the cysteine residues indeed are important for the role of YjbH in disulfide stress management. A possible difference underlying the obtained results is the stability of the mutated YjbH proteins.…”

The Staphylococcus aureus Thiol/Oxidative Stress Global Regulator Spx Controls trfA , a Gene Implicated in Cell Wall Antibiotic Resistance

“…Our observation that Spx protein levels were dramatically stabilized using oxacillin as a stimulus whereas spx transcription was unaffected strongly suggests that cell wall antibiotic stress acts on Spx primarily at the posttranscriptional level. Redox regulation of cysteine residues in YjbH has been proposed as a mechanism governing the proteolytic turnover of Spx (40,57). In this scenario, oxidation of cysteines would have the dual effect of disrupting YjbH-Spx interaction, as well as possibly promoting Spx-␣CTD interaction through oxidation of the Spx cysteine switch (58).…”

“…Loss of YjbH, a negative regulator and interacting partner of Spx, has been linked to reduced sensitivity to diamide (38) in B. subtilis, as well as nitrosative stress via altered susceptibility to sodium nitroprusside (56). In S. aureus, disruption of the corresponding YjbH ortholog results in pleiotropic effects that include altered sensitivity to ␤-lactam and glycopeptide antibiotics (10,37,57), as well as enhanced peptidoglycan cross-linking and overproduction of penicillin binding protein PBP4 (57).…”

The Staphylococcus aureus Chaperone PrsA Is a New Auxiliary Factor of Oxacillin Resistance Affecting Penicillin-Binding Protein 2A

“…Membrane proteins were isolated as previously described (25). Strains were grown in TSB until midexponential phase and pellets were resuspended in 1/250 of the initial culture volume of buffer A (50 mM KPO 4 buffer [pH 7.4], 10 mM MgCl 2 ).…”